PROJECT SUMMARY The ESX-1 (ESAT-6 system-1) secretion system is essential for mycobacterial pathogenesis. Yet, the specific role of the ESX-1 secreted proteins (substrates) in protein secretion and virulence remains unclear. Because the inactivation of individual ESX-1 substrate genes reportedly results in the same phenotype, genetic approaches have provided little information regarding the functional roles of ESX-1 substrates. Without understanding the contribution of ESX-1 substrates, the field faces a major hurdle in understanding the mechanisms underlying ESX-1-mediated secretion and virulence. The applicant's long-term goal is to understand the molecular mechanisms underlying mycobacterial protein secretion. The objective of this proposal is to uncover the functional relationships between ESX-1 substrates. The rationale underlying the proposal is that the successful completion of the proposed research will provide basic insight into the relationship between ESX-1 substrates, providing a new framework for understanding ESX-1 secretion. The applicant's lab has uncovered that the deletion of individual ESX-1 substrate genes results in discrete phenotypes that have been previously missed. The central hypothesis of the proposal is that by leveraging the unique secretory phenotypes associated with the deletion of individual and pairs of genes encoding substrates, fundamental but elusive aspects of ESX-1 protein secretion can be defined. The central hypothesis will be tested by these specific aims: 1). Define the requirement of each substrate in ESX-1 secretion. 2) Define the functional relationship between ESX-1 substrates. Under Aim 1, the applicant proposes a quantitative proteomics approach to measure the changes in ESX-1-mediated protein secretion due to the loss of individual ESX-1 substrates. Under Aim 2, the applicant proposes to define genetic relationships between ESX-1 substrates by measuring how pairwise deletion of ESX- 1 substrate genes impacts mycobacterial secretion and virulence. Understanding how individual substrates promote ESX-1 secretion and virulence is significant because it will open new avenues for investigation into how each ESX-1 substrate promotes secretion and virulence, which could inform the treatment and prevention of mycobacterial infections. The proposed research is innovative because it challenges the status quo by providing a means to define discrete phenotypes of and functional relationships between ESX-1 substrates. Collectively, the completion of both aims will advance the field vertically by defining fundamental but elusive features of the mechanistic biology underlying ESX-1 protein secretion.